Plxnd1 expression in thymocytes regulates their intrathymic migration while that in thymic endothelium impacts medullary topology

Abstract

An important role for plexinD1 in thymic development is inferred from studies of germline Plxnd1 knockout (KO) mice where mislocalized CD69(+) thymocytes as well as ectopic thymic subcapsular medullary structures were observed. Given embryonic lethality of the Plxnd1 (-/-) genotype, fetal liver transplantation was employed in these prior analyses. Such embryonic hematopoietic reconstitution may have transferred Plxnd1 KO endothelial and/or epithelial stem cells in addition to Plxnd1 KO lymphoid progenitors, thereby contributing to that phenotype. Here we use Plxnd1 (flox/flox) mice crossed to pLck-Cre, pKeratin14-Cre, or pTek-Cre transgenic animals to create cell-type specific conditional knockout (CKO) lines involving thymocytes (D1ThyCKO), thymic epithelium (D1EpCKO), and thymic endothelium (D1EnCKO), respectively. These CKOs allowed us to directly assess the role of plexinD1 in each lineage. Loss of plexinD1 expression on double positive (DP) thymocytes leads to their aberrant migration and cortical retention after TCR-mediated positive selection. In contrast, ectopic medulla formation is a consequence of loss of plexinD1 expression on endothelial cells, in turn linked to dysregulation of thymic angiogenesis. D1EpCKO thymi manifest neither abnormality. Collectively, our findings underscore the non-redundant roles for plexinD1 on thymocytes and endothelium, including the dynamic nature of medulla formation resulting from crosstalk between these thymic cellular components.

Keywords: angiogenesis; autoimmunity; plexind1; thymic development; thymic epithelial cells.

Figure 1

PlexinD1 is not expressed in D1ThyCKO thymocytes but developmental progression is normal. (A) PlexinD1 was detected in total thymocyte lysates from 3- to 4-week-old WT or D1ThyCKO mice (three mice/strain) by Western blotting. The lower bands (**) are non-specific bands common to both WT and D1ThyCKO thymocyte extracts and do not represent plexinD1 (representative of four independent experiments). (B) PlexinD1 detected by binding of sema3E-Fc (blue line) or control IgG2c (red line) after gating DP, CD4SP, CD8SP, and DN populations based on CD4 and CD8α expression (representative of six independent experiments). (C) Total thymocytes/thymus from WT or D1ThyCKO animals (n = 10 for each strain; P = 0.737, not significant). (D) Thymocyte differentiation in WT and D1ThyCKO mice. The percentage representations of DN (CD4⁻CD8⁻), DP (CD4⁺CD8⁺), CD4SP (CD4⁺CD8⁻), and CD8SP (CD4⁻CD8⁺) are depicted (upper panels). The percentages of DN1 (CD44^hiCD25^lo), DN2 (CD44^hiCD25^hi), DN3 (CD44^loCD25^hi), and DN4 (CD44^loCD25^lo) are shown (lower panels). Results represent six independent experiments.

Figure 2

CD69⁺ thymocytes are retained in the cortex in D1ThyCKO and Sema3e^−/− thymus but track to the medulla in WT and D1EpCKO mice. For each strain, thymic frozen sections were stained for CD4 (green) and CD8 (red) expression where the DP cells in the cortical regions appear yellow in merged images due to the co-expression of CD4 and CD8. The merged yellow DP signal was used to delineate the cortex (C) from the individual SP red and green signals in the medulla (M) and a yellow dashed line is inserted to indicate the apparent corticomedullary junction. A second yellow dashed line indicates the capsule position. CD69 is an activation marker expressed by positively selected thymocytes following TCR stimulation. ER-TR5 is considered a medullary marker (29) while MHCII (I-A/I-E) is preferentially expressed in medulla (30). Each figure is representative of three independent experiments. White bar in the merged images is 100 μm.

Figure 3

Loss of plexinD1 disrupts cortical to medullary translocation of CD69⁺ DP thymocytes. (A) The regional distribution of CD69⁺ cells in five separate frozen sections for each strain was determined based on FITC signal intensity within the regions defined by the apparent corticomedullary junction. After subtracting background signal, the ratio of fluorescence signal strength between the cortex and the medulla was calculated. A value >1 indicates stronger medullary presence on comparison with cortex. Error bars represent SEM. (B) Medullary structures were visualized by hematoxylin and eosin (H&E) staining of WT, D1ThyCKO, and D1EpCKO thymi as well as in thymus in irradiated animals repopulated with Plxnd1^−/− fetal liver. The red arrows in the Plxnd1^−/− section indicate regions of fused thymic medulla and capsule. Each figure is representative of three independent experiments.

Figure 4

pre-TCR DP thymocytes can induce medullary formation on activation. At time points from 0 h to 3 weeks, cryosections from thymi of Tcrb^+/+Rag2^−/− mice injected at 0 h with anti-CD3ε mAb were prepared. Adjacent cryosections were stained with anti-CD69 to identify activated DP thymocytes, anti-CD4, and anti-CD8 to detect DP and SP thymocytes and differentiate cortical from developing medullary structures, and with ER-TR5, UEA1, and anti-MHCII (I-A/I-E) to identify putative medullary elements. Five slides of each thymus were H&E stained. This experiment was repeated with identical results. White bar in the merged images represents 100 μm and the black bar in the H&E panels represent 5 mm.

Figure 5

PlexinD1 on thymic endothelial cells regulates the distribution of thymic medullary formations. (A) Schematic for analysis of D1EnCKO cells on thymus development. Fetal thymi harboring WT (both fused lobes transplanted) or D1EnCKO (single lobe transplanted) endothelial cells were allowed to develop under the recipient’s renal capsule. All cell populations in the transplanted D1EnCKO thymus other than endothelial cells expressed plexinD1 appropriately as did all cells in the normal recipients. (B) Red arrows in H&E stained panels indicate regions of medulla fused with the thymic capsule. A frozen section corresponding to the inset region in the D1EnCKO panel was stained for keratin 8 highly represented in cTEC using the Troma1 antibody (56, 57) and for ER-TR5 representing medullary development. Cortical-medullary demarcation was confirmed by the relative representation of merged CD4⁺CD8⁺ signal for DP cells and SP CD4 and CD8 cell development in the subcapsular region. Result is representative of three independent transplantation experiments. White bar in the confocal image represents 100 μm and in the H&E stained image represents 5 mm. (C) Cryosections of WT and D1EnCKO thymus were stained with anti-CD4-APC, anti-CD8α-TRITC, and anti-ESAM mAb-FITC. Dashed yellow line follows the corticomedullary junction; in the D1EnCKO panel, a second dashed yellow line to the left indicates the ESAM⁺ cells in the subcapsullar zone. C, cortex; M, medulla. Result is representative of three independent transplantation experiments. White bar in the merged images are 100 μm. (D) Pecam1 staining to identify developing vascular endothelial structures. High density nuclear staining by DAPI delineates cortical regions and lower density nuclear staining correlates with medullary structures. C, cortex; M, medulla. The corticomedullary junctions and capsules are identified by dashed yellow lines. White bar in the images is 5 mm.

Figure 6

Differential development of ESAM⁺ blood vessels in WT, D1EnCKO and Sema3e^−/− thymi. Thymic cryosections of the indicated control mice and D1EnCKO (A) and Sema3e^−/− (B) mice were stained with anti-MHCII-APC, anti-CD8α-TRITC, and anti-ESAM mAb-FITC. Dashed line follows the corticomedullary junction. The result is representative of one of three independent experiments. The black bar below each merged image represents 100 μm and is applicable to every panel in each block. C, cortex; M, medulla.